The objective of these studies is to delineate the molecular mechanisms by which brain Ca++/calmodulin(Cam)-dependent protein kinase II (CaM-kinase II) is regulated by binding of Ca /CaM and by autophosphorylation, both with the purified kinase and in intact cells and tissues. Long-term regulation of brain CaM-kinase II by covalent mechanisms (i.e., autophosphorylation) is of special interest because of the putative neuronal functions of this kinase. There is good evidence that presynaptically CaM-kinase II is intimately involved in Ca-dependent regulation of catecholamine biosynthesis and neurotransmitter exocytosis. CaM-kinase II comprises 50% of protein in the postsynaptic density of excitatory synapses and may be involved in long-term alterations of synaptic plasticity. Previous studies from this and other laboratories have led to the formulation of a model for the regulation of CaM-kinase II by binding of Ca/CaM and by autophosphorylation. This model will be tested using synthetic peptides, derived from the known sequence of the 50 kDa subunit of the brain CaM-kinase II, to characterize CaM-binding and substrate-directed inhibitory domains within the kinase sequence. Amino acids essential to these functions will be identified using altered peptide sequences. The effects of phosphorylation of consensus phosphorylation sites flanking these two domains will be assessed in terms of the function of these domains. These results will be utilized in the design of in vitro site-specific mutagenesis to test the functions of specific amino acids on the properties of the kinase. Lastly the ability of CaM- kinase II to undergo autophosphorylation and formation of Ca independent species in intact cells will be assessed in hippocampal brain slices and in cultured hippocampal cells and PC12 cells in response to Ca influx. These studies will further our understanding of the regulation and function of this multifunctional Ca++ -dependent kinase.